This invention relates to a new class of chemical compounds and to their use in medicine. In particular, the invention concerns novel amide derivatives, methods for their preparation, pharmaceutical compositions containing them and their use as thrombin inhibitors.
Thrombin inhibitors have been described previously in International Patent Application No. WO97/22589.
Thrombin is a serine proteinase present in plasma and is formed by conversion from its prothrombin precursor by the action of Factor Xa. Thrombin plays a central role in the mechanism of blood coagulation by converting the soluble plasma protein, fibrinogen, into insoluble fibrin. The insoluble fibrin matrix is required for the stabilisation of the primary hemostatic plug. Many significant disease states are related to abnormal hemostasis. With respect to the coronary arterial vasculature, abnormal thrombus formation due to the rupture of an established atherosclerotic plaque is the major cause of acute myocardial infarction and unstable angina. Both treatment of an occlusive coronary thrombus by thrombolytic therapy and percutaneous transluminal coronary angioplasty (PTCA) are often accompanied by an acute thrombotic reclosure of the affected vessel which requires immediate resolution. With respect to the venous vasculature, a high percentage of patients undergoing major surgery in the lower extremities or the abdominal area suffer from thrombus formation in the venous vasculature which can result in reduced blood flow to the affected extremity and a pre-disposition to pulmonary embolism. Disseminated intravascular coagulopathy commonly occurs within both vascular systems during septic shock, certain viral infections and cancer and is characterised by the rapid consumption of coagulation factors and systemic coagulation which results in the formation of life-threatening thrombi occurring throughout the vasculature leading to widespread organ failure.
Beyond its direct role in the formation of fibrin rich blood clots, thrombin has been reported to have profound bioregulatory effects on a number of cellular components within the vasculature and blood, (Shuman, M. A., Ann. NY Acad. Sci., 405:349 (1986)).
The inhibition of thrombin has been implicated as a potential treatment for a number of disease states. Thrombin inhibitors may be useful in the treatment of acute vascular diseases such as coronary thrombosis, stroke, pulmonary embolism, deep vein thrombosis, restenosis, atrial fibrillation, myocardial infarction, and unstable angina. They have been described as anti-coagulant agents both in-vivo and ex-vivo, and in oedema and inflammation, whereby a low dose of thrombin inhibitor can reduce platelet and endothelial cell thrombin mediated inflammatory responses without concomitant anticoagulant effects. Thrombin has been reported to contribute to lung fibroblast proliferation, thus, thrombin inhibitors could be useful for the treatment of some pulmonary fibrotic diseases. Thrombin inhibitors have also been reported in the treatment of tumour metastasis whereby the thrombin inhibitor prevents the fibrin deposition and metastasis caused by the inappropriate activation of Factor X by cysteine proteinases produced by certain tumour cells. They have been shown to inhibit neurite retraction and thus may have potential in neurogenerative diseases such as Parkinson""s and Alzheimer""s disease. They have also been reported to be used in conjunction with thrombolytic agents by permitting the use of a lower dose of thrombolytic agent. Other potential uses have been described in U.S. Pat. No. 5,371,091 for the treatment of Kasabach Merritt Syndrome and haemolytic uremic syndrome, in EP565897 for the prevention of fibrin deposits in the eye during ophthalmic surgery, and in DE4126277 for the treatment of osteoporosis.
Thus, we have now found a novel class of amide derivatives which act as thrombin inhibitors shown as formula (I) 
where
R1 represents C1-4alkyl or C3-8cycloalkyl;
R2 represents C1-4alkyl or C3-4alkenyl;
R3 represents hydrogen, C1-3alkyl or halogen;
R4 represents C1-6alkyl;
and pharmaceutically acceptable derivatives or solvates thereof.
Referring to the general formula (I), alkyl includes both straight and branched chain saturated hydrocarbon groups, e.g. methyl, ethyl and isopropyl; cycloalkyl includes saturated cyclic hydrocarbon groups, e.g. cyclopentyl and cyclohexyl; alkenyl includes both straight and branched chain hydrocarbon groups containing one double bond, e.g. propenyl, 2-methylpropenyl and butenyl.
It will be appreciated that a compound of formula (I) contains a chiral centre at the position denoted by *. Thus, each compound within formula (I) may exist in two distinct optical isomeric forms. The scope of the present invention extends to cover individual enantiomers of compounds of formula (I) and mixtures of enantiomers of compounds of formula (I) in any proportion, including racemic mixtures. Generally it is preferred to use a compound of formula (I) in the form of a purified single enantiomer, most preferably the (S) isomer.
Referring to general formula (I), R1 suitably represents propyl, isopropyl, butyl, cyclopentyl or cyclohexyl. R1 is preferably isopropyl.
R2 is suitably methyl, ethyl, propyl or isopropyl. R2 is preferably ethyl.
R3 is suitably methyl or chloro. R3 is preferably methyl.
R4 is suitably methyl or ethyl. R4 is preferably methyl.
Suitable compounds of general formula (I) for use according to the invention include:
N-Ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
N,N-Diisopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
N-Isopropyl-3,N-dimethyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
3,N-Dimethyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
3-Methyl-N,N-dipropyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
N-Ethyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
N-Butyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
N-Cyclohexyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
N-Isopropyl-3-methyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
3-Chloro-N-isopropyl-N-propyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide;
3-Chloro-N,N-diisopropyl-5-[2-(pyridin-4-ylamino)-butoxy]-benzamide;
and pharmaceutically acceptable derivatives or solvates thereof.
Particular compounds of general formula (I) for use according to the invention include:
N-Ethyl-N-isopropyl-3-methyl-5-[2S-(pyridin-4-ylamino)-propoxy]-benzamide; and pharmaceutically acceptable derivatives or solvates thereof.
By xe2x80x9ca pharmaceutically acceptable derivativexe2x80x9d is meant any pharmaceutically acceptable salt, or a metabolically labile derivative, for example a derivative of an amine group, of a compound of formula (I) or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof. It will be appreciated by those skilled in the art that the compounds of formula (I) may be modified to provide pharmaceutically acceptable derivatives thereof at any of the functional groups in the compounds of formula (I). Such derivatives are clear to those skilled in the art, without undue experimentation, and with reference to the teaching of Burger""s Medicinal Chemistry And Drug Discovery, 5th Edition, Vol 1: Principles And Practice, which is incorporated herein by reference.
Preferred pharmaceutically acceptable derivatives of the compounds of formula (I) are pharmaceutically acceptable salts thereof.
Pharmaceutically acceptable salts of the compounds of formula (I) include those derived from pharmaceutically acceptable inorganic and organic acids. Examples of suitable acids include hydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicylic, succinic, toluene-p-sulphonic, di-p-toluoyl tartrate, sulfanilic, tartaric, acetic, citric, methanesulphonic, formic, benzoic, malonic, naphthalene-2-sulphonic and benzenesulphonic acids. Preferred pharmaceutically acceptable salts of the compounds of formula (I) include the toluene-p-sulphonic acid salt. Other acids such as oxalic, while not in themselves pharmaceutically acceptable may be useful in the preparation of salts useful as intermediates in obtaining compounds of the invention and their pharmaceutically acceptable acid addition salts.
The suitability of compounds of formula (I) as thrombin inhibitors is exhibited by their ability to inhibit human xcex1-thrombin in a chromogenic assay, using N-p-tosyl-gly-pro-lys p-nitroanilide as the chromogenic substrate.
Furthermore, the compounds of formula (I) exhibit effective anti-coagulant activity in vitro as indicated by the APTT assays herein described.
Furthermore, the compounds of formula (I) exhibit effective anti-thrombotic activity as indicated in the Arterio-Venous Shunt Model herein described.
Thus, the compounds of formula (I) are useful in the treatment of clinical conditions susceptible to amelioration by administration of a thrombin inhibitor. Such conditions include acute vascular diseases such as coronary thrombosis, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, restenosis, and atrial fibrillation; in oedema and PAF mediated inflammatory diseases such as adult respiratory shock syndrome, septic shock and reperfusion damage; the treatment of pulmonary fibrosis; the treatment of tumour metastasis; neurogenerative disease such as Parkinson""s and Alzheimer""s diseases; viral infection; Kasabach Merritt Syndrome; haemolytic uremic syndrome; arthritis; osteoporosis; as anti-coagulants for extracorporeal blood in for example, dialysis, blood filtration, bypass, and blood product storage; and in the coating of invasive devices such as prostheses, artificial valves and catheters in reducing the risk of thrombus formation.
Accordingly, the present invention provides a method of treatment of a mammal, including man, suffering from conditions susceptible to amelioration by a thrombin inhibitor which method comprises administering to the subject an effective amount of a compound of general formula (I) or a pharmaceutically acceptable derivative thereof.
References in this specification to treatment include prophylactic treatment as well as the alleviation of symptoms.
In a further aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use as a therapeutic agent for use in medicine, particularly human medicine.
In a further aspect, the invention provides the use of a compound of general formula (I) or a pharmaceutically acceptable derivative thereof, for the manufacture of a medicament for the treatment of a condition susceptible to amelioration by a thrombin inhibitor.
While it is possible that, for use in therapy, a compound of the invention may be administered as the raw chemical it is preferable to present the active ingredient as a pharmaceutical formulation.
The invention thus further provides a pharmaceutical formulation comprising a compound of formula (I) or a pharmaceutically acceptable derivative thereof together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The compounds of the present invention may be used in combination with other antithrombotic drugs such as thromboxane receptor antagonists, prostacyclin mimetics, phosphodiesterase inhibitors, fibrinogen antagonists, thrombolytic drugs such as tissue plaminogen activator and streptokinase, non-steroidal anti-inflammatory drugs such as aspirin, and the like.
Thus the compounds for use according to the present invention may be formulated for oral, buccal, parenteral, topical, rectal, or transdermal administration or in a form suitable for administration by inhalation or insufflation (either through the mouth or the nose).
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycollate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
The compounds according to the present invention may be formulated for parenteral administration by injection e.g. by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
The compounds according to the present invention may be formulated for topical administration by insufflation and inhalation. Examples of types of preparation for topical administration include sprays and aerosols for use in an inhaler or insufflator, or a formulated powder for use in an inhaler.
Powders for external application may be formed with the aid of any suitable powder base, for example, lactose, talc, or starch. Spray compositions may be formulated as aqueous solutions or suspensions or as aerosols delivered from pressurised packs, such as metered dose inhalers, with the use of a suitable propellant.
The compounds according to the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously, transcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds according to the present invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A proposed dose of the compounds according to the present invention for administration to a human (of approximately 70 kg body weight) is 0.1 mg to 1 g, preferably to 1 mg to 500 mg of the active ingredient per unit dose, expressed as the weight of free base. The unit dose may be administered, for example, 1 to 4 times per day. The dose will depend on the route of administration. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the patient as well as the severity of the condition to be treated. The precise dose and route of administration will ultimately be at the discretion of the attendant physician or veterinarian.
The compounds of the invention may be prepared by any of the processes known in the art for the preparation of similar compounds. For example, according to a first process (A) wherein R1, R2, R3, and R4, are as previously defined, compounds of formula (I) may be prepared by deprotection of a compound of formula (II), 
where P1 represents a suitable protecting group such as tert-butoxycarbonyl, under suitable conditions, e.g. acidic conditions for the removal of a tert-butoxycarbonyl group.
According to a second process, (B), a compound of formula (I) may be prepared by reaction of a compound of formula (III) with a compound of formula (IV) 
where R7 represents hydrogen, and L represents hydroxyl. The coupling is conveniently carried out using standard reagents such as diethyl azodicarboxylate and triphenylphosphine in a suitable solvent such as toluene.
According to a third process, (C), a compound of formula (I) may be prepared by reaction of a compound of formula (III) with a compound of formula (IV) where R7 represents hydrogen, and L represents a suitable leaving group, such as chloride, in the presence of a suitable base, such as potassium carbonate. The coupling is conveniently effected in a suitable solvent such as N,N-dimethylformamide, preferably at elevated temperature.
According to a fourth process, (D), a compound of formula (I) may be prepared from reaction of compounds of formula (V) and formula (VI), 
where R7 represents hydrogen. The reaction may be conveniently carried out in the presence of an activating agent or agents such as 1-hydroxybenzotriazole, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and a base such as ethyidiisopropylamine in a suitable solvent such as N,N-dimethylformamide.
A compound of formula (II) may be prepared by reaction of a compound of formula (III) with a compound of formula (IV) where R7 represents P1, as defined above, and L represents hydroxyl or a suitable leaving group such as 4-toluenesulfonate (tosylate). Where L represents hydroxyl the coupling is conveniently carried out using conditions as similarly used for process (B). Where L represents tosylate the coupling is conveniently carried out in a suitable solvent such as N,N-dimethylformamide in the presence of a suitable base such as sodium hydride.
A compound of formula (II) may also be prepared by reaction of compounds of formula (V) and formula (VI) where R7 represents P1 as defined above, suitably using the conditions of process (D).
Compounds of formula (III) may be prepared from compounds of formula (VII) 
conveniently using boron tribromide in a suitable solvent such as dichloromethane.
Compounds of formula (III) may also be prepared from compounds of formula (IX) 
conveniently by reaction with an acid chloride such as pivaloyl chloride, in the presence of a base such as triethylamine, in a suitable solvent such as toluene, followed by reaction with compounds of formula (VI).
Compounds of formula (VII) may be prepared by reaction of compounds of formula (VII) and formula (VI) 
conveniently according to the conditions of process (D). Alternatively, the reaction of compounds of formula (VIII) and formula (VI) may be carried out using oxalyl chloride in the presence of N,N-dimethylformamide in a suitable solvent such as tetrahydrofuran.
Compounds of formula (V) may be prepared by oxidation of the corresponding aldehyde of formula (X) 
where R7 represents hydrogen or P1. The conversion is effected by treatment of the aldehyde with a suitable oxidising agent such as sodium chlorite in the presence of sulfamic acid in a mixture of water and 1,4-dioxan.
Compounds of formula (X) may be prepared from compounds of formula (XI) and (IV) 
where R7 represents hydrogen or P1 and L represents hydroxyl or a suitable leaving group such as 4-toluenesulfonate (tosylate), providing that where L is a suitable leaving group, R7 preferably represents P1. Where L represents hydroxyl the coupling is carried out using standard reagents identical to those employed in process (B). Where L represents tosylate the coupling is carried in a suitable solvent such as N,N-dimethylformamide in the presence of a suitable base such as sodium hydride.
Compounds of formula (V) may also be prepared from compounds of formula (XII), 
where R7 represents hydrogen or P1 and R8 represents a suitable protecting group such as alkyl, e.g. methyl. The reaction is carried out using appropriate conditions such as lithium hydroxide in 1,4-dioxan or aqueous sodium hydroxide in ethanol.
Compounds of formula (XII) may be prepared from reaction of compounds of formula (XIII) and (IV) 
where R7 represents hydrogen or P1, R8 represents a suitable protecting group such as alkyl, e.g. methyl, and L represents hydroxyl or a suitable leaving group such as 4-toluenesulfonate (tosylate), providing that where L is a suitable leaving group, R7 preferably represents P1, using suitable conditions similar to those employed for the synthesis of compounds of formula (X).
Compounds of formula (IV), (VI), (VIII), (IX), (XI), and (XIII) are known in the art or may be prepared by standard methods as herein described.
The compounds of the invention possess thrombin inhibitory activity as determined in vitro by their ability to inhibit human xcex1-thrombin in a chromogenic assay, using N-p-tosyl-gly-pro-lys p-nitroanilide as the chromogenic substrate. All dilutions were made in a buffer consisting of: 50 mM HEPES, 150 mM NaCl, 5 mM CaCl2 0.1% PEG and at pH 7.4. Briefly, the substrate (final conc. of 100 xcexcM) was added to thrombin (final conc. of 1 nM) and the reaction monitored for 10 mins at 405 nm using a Biotek EL340 plate reader; the assay was performed at room temperature. To obtain IC50s the data were analyzed using Kineticalc(trademark) and processed using ActivityBase(trademark) to obtain the IC50 value. To determine the IC50 at zero and 15 mins. the compounds were preincubated with thrombin for these times prior to adding the chromogenic substrate.
The compounds of the invention possess anti-coagulant activity as determined in vitro by their ability to extend the clotting time of human plasma, the activated partial thromboplastin time (APTT). Pooled citrated (0.38% trisodium citrate w/v) plasma was prepared from blood taken from healthy volunteers and stored at xe2x88x9270xc2x0 C. The APTT tests were performed using a Thrombtrack 4 from Nycomed. Actin reagent (a reconstituted extract from dehydrated rabbit brain, also containing ellagic acid) was obtained from Baxter Healthcare Corporation USA. Briefly, citrated plasma was added to either compound or distilled water followed by addition of actin reagent. These were then mixed for 2 min at 37xc2x0 C. before adding calcium chloride to initiate clotting. Compounds extended the normal clotting time, which is in the range 30-35 seconds, to varying degrees depending on their concentrations. The degrees of extension of the APTT was calculated by the ratio of clotting times in presence or absence of compound. The concentration of a compound to extend the xe2x80x98normalxe2x80x99 APTT by 1.5xc3x97 was used as a criterion for comparing the anti-coagulant activities of compounds.
The results below illustrate the thrombin inhibitory activity and the anti-coagulant activity of a range of compounds of formula (I) using the above described biological methods:
The compounds of the invention possess anti-thrombotic activity as determined in vivo by their ability to reduce thrombus formation in a rat arterio-venous shunt model. Anaesthetised (Inactin 120 mg/kg i.p.) rats were prepared by the insertion of an extracorporeal shunt between the left carotid artery and the right jugular vein. The shunt consisted of two 12 cm lengths of polythene tubing (Portex; 0.58 and 0.86 mm internal diameter respectively) connected by 3 mm (base diameter) silicone rubber bungs (Jencons Scientific Ltd) to a 6 cm length of polythene tubing (Portex; 3 mm internal diameter). The tubing was connected via drilled holes through the centre of each bung. An 8 cm piece of silk thread was held taut between the two bungs, passing through the central holes, so that it remained longitudinally orientated in the central portion of the shunt. Before cannulation the shunt was filled with 154 mM sodium chloride solution (saline).
Following cannulation a haemostatic clip was left in position on the carotid artery to prevent blood flow through the shunt. The left carotid artery was also instrumented with an ultrasonic flow probe (Transonic Systems Inc., 0.5 mm) which was connected to a Transonic flow meter (model T206) for the continuous display of phasic carotid artery blood flow. Continuous carotid artery blood flow was acquired by an MI2 data acquisition system (Modular Instruments Inc.).
Following shunt cannulation, and an equilibration period, the protocol was commenced by the administration of vehicle or compound. The pre-treatment time was 30 min and was followed by the removal of the haemostatic clip from the carotid artery thus allowing blood flow through the shunt. Following 15 min of shunt blood flow, the arterial clip was replaced, the shunt removed, and 0.5 ml saline injected slowly through the central portion of the shunt to remove free blood. The cotton thread, and associated thrombus, was carefully removed and the weight of the thrombus determined. Coagulation parameters, including activated partial thromboplastin time (APTT), were calculated. A 2 ml blood sample was taken by direct cardiac puncture and transferred to a tube containing trisodium citrate (ratio 9:1, final concentration of citrate 12.9 mM). The blood sample was mixed gently and transferred to eppendorf tubes and centrifuged at 10000 g for 2 min. The plasma was decanted and stored at 4xc2x0 C. until analysis. All tests were performed on a Sysmex CA5000 automated analyser according to the instruction manual.
Antithrombotic activity was assessed by a decrease in thrombus weight, an extension in the time to occlusion, and an increase in the blood flow area, and was related to effects upon the coagulation parameters measured.
The invention is further illustrated by the following intermediates and examples.
H.p.l.c. high performance liquid chromatography
Rt retention time
DIPEA N-ethyidiisopropylamine
DMF N,N-dimethylformamide
TBTU 2-(1 H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
DMAP 4-dimethylaminopynidine
br broad
s singlet
d doublet
t triplet
m multiplet
t.l.c. thin layer chromatography
Analytical H.p.l.c. was carried out on a Hewlett Packard Series II 1090 Liquid Chromatograph using a Rainin Microsorb C18 column (size 4.6xc3x97150 mm, catalog number 80-215-C5) operating at a flow rate of 1.5 ml/min. Eluents were A: 0.1% trifluoroacetic acid/water, B: 0.05% trifluoroacetic acid/acetonitrile. Gradients:
System 1:15-95% B in A over 15 min
Retention times are given for a wavelength (xcex) of 254 nm unless otherwise stated.
Preparative H.p.l.c.:
System A: Supelcosil LC-ABZ column (size 21.2 mmxc3x9725 cm or 21.2 mmxc3x9710 cm) operating at 15 ml/min (eluents were A: 0.1% trifluoroacetic acid/water, B: 0.01% trifluoroacetic acid in 95:5 acetonitrile/water).
System B: 50 mm Prochrom column packed with 200 g Sorbsil C60 silica gel operating at 80 mls/min [eluent was:dichloromethane (80), methanol (20), acetic acid (0.5) and ammonia (0.5)].
T.l.c. was carried out using Camiab silica (Polygram(copyright) SILG/UV254). Eluent was dichloromethane:ethanol:aqueous ammonia in stated ratio.
Flash column chromatography was carried out on Merck silica gel (Merck 9385) or using SI Megabond Elut(copyright) (normal bonded phase, size 60 cc/10 g) cartridges.